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Integrated nonlinear optical imaging microscope for on-axis crystal detection and centering at a synchrotron beamline.

ABSTRACT: Nonlinear optical (NLO) instrumentation has been integrated with synchrotron X-ray diffraction (XRD) for combined single-platform analysis, initially targeting applications for automated crystal centering. Second-harmonic-generation microscopy and two-photon-excited ultraviolet fluorescence microscopy were evaluated for crystal detection and assessed by X-ray raster scanning. Two optical designs were constructed and characterized; one positioned downstream of the sample and one integrated into the upstream optical path of the diffractometer. Both instruments enabled protein crystal identification with integration times between 80 and 150 µs per pixel, representing a ?10(3)-10(4)-fold reduction in the per-pixel exposure time relative to X-ray raster scanning. Quantitative centering and analysis of phenylalanine hydroxylase from Chromobacterium violaceum cPAH, Trichinella spiralis deubiquitinating enzyme TsUCH37, human ?-opioid receptor complex kOR-T4L produced in lipidic cubic phase (LCP), intimin prepared in LCP, and ?-cellulose samples were performed by collecting multiple NLO images. The crystalline samples were characterized by single-crystal diffraction patterns, while ?-cellulose was characterized by fiber diffraction. Good agreement was observed between the sample positions identified by NLO and XRD raster measurements for all samples studied.

SUBMITTER: Madden JT

PROVIDER: S-EPMC3682636 | biostudies-literature | 2013 Jul

REPOSITORIES: biostudies-literature

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Integrated nonlinear optical imaging microscope for on-axis crystal detection and centering at a synchrotron beamline.

Madden Jeremy T JT Toth Scott J SJ Dettmar Christopher M CM Newman Justin A JA Oglesbee Robert A RA Hedderich Hartmut G HG Everly R Michael RM Becker Michael M Ronau Judith A JA Buchanan Susan K SK Cherezov Vadim V Morrow Marie E ME Xu Shenglan S Ferguson Dale D Makarov Oleg O Das Chittaranjan C Fischetti Robert R Simpson Garth J GJ

Journal of synchrotron radiation 20130503 Pt 4

Nonlinear optical (NLO) instrumentation has been integrated with synchrotron X-ray diffraction (XRD) for combined single-platform analysis, initially targeting applications for automated crystal centering. Second-harmonic-generation microscopy and two-photon-excited ultraviolet fluorescence microscopy were evaluated for crystal detection and assessed by X-ray raster scanning. Two optical designs were constructed and characterized; one positioned downstream of the sample and one integrated into t ...[more]

PMID: 23765294

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Project description:PURPOSE:To characterize the first in-human intraoperative imaging using a custom prototype spectral-domain microscope-integrated optical coherence tomography (MIOCT) device during vitreoretinal surgery with instruments in the eye. METHODS:Under institutional review board approval for a prospective intraoperative study, MIOCT images were obtained at surgical pauses with instruments held static in the vitreous cavity and then concurrently with surgical maneuvers. Postoperatively, MIOCT images obtained at surgical pauses were compared with images obtained with a high-resolution handheld spectral-domain OCT (HHOCT) system with objective endpoints, including acquisition of images acceptable for analysis and identification of predefined macular morphologic or pathologic features. RESULTS:Human MIOCT images were successfully obtained before incision and during pauses in surgical maneuvers. MIOCT imaging confirmed preoperative diagnoses, such as epiretinal membrane, full-thickness macular hole, and vitreomacular traction and demonstrated successful achievement of surgical goals. MIOCT and HHOCT images obtained at surgical pauses in two cohorts of five patients were comparable with greater than or equal to 80% correlation in 80% of patients. Real-time video-imaging concurrent with surgical manipulations enabled, for the first time using this device, visualization of dynamic instrument-retina interaction with targeted OCT tracking. CONCLUSION:MIOCT is successful for imaging at surgical pauses and for real-time image guidance with implementation of targeted OCT tracking. Even faster acquisition speeds are currently being developed with incorporation of a swept-source MIOCT engine. Further refinements and investigations will be directed toward continued integration for real-time volumetric imaging of surgical maneuvers. TRANSLATIONAL RELEVANCE:Ongoing development of seamless MIOCT systems will likely transform surgical visualization, approaches, and decision-making.

Project description:PurposeWe develop and assess the impact of depth-based, motion-stabilized colorization (color) of microscope-integrated optical coherence tomography (MIOCT) volumes on microsurgical performance and ability to interpret surgical volumes.MethodsColor was applied in real-time as gradients indicating axial position and stabilized based on calculated center of mass. In a test comparing colorization versus grayscale visualizations of prerecorded intraoperative volumes from human surgery, ophthalmologists (N = 7) were asked to identify retinal membranes, the presence of an instrument, its contact with tissue, and associated deformation of the retina. In a separate controlled trial, trainees (N = 15) performed microsurgical skills without conventional optical visualization and compared colorized versus grayscale MIOCT visualization on a stereoptic screen. Skills included thickness identification, instrument placement, and object manipulation, and were assessed based on time, performance metrics, and confidence.ResultsIn intraoperative volume testing, colorization improved ability to differentiate membrane from retina (P < 0.01), correctly identify instrument contact with membrane (P = 0.03), and retinal deformation (P = 0.01). In model microsurgical skills testing, trainees working with colorized volumes were faster (P < 0.01) and more correct (P < 0.01) in assessments of thickness for recessed and elevated objects, were less likely to inadvertently contact a surface when approaching with an instrument (P < 0.01), and uniformly more confident (P < 0.01 for each) in conducting each skill.ConclusionsDepth-based colorization enables effective identification of retinal membranes and tissue deformation. In microsurgical skill testing, it improves user efficiency, and confidence in microscope-independent, OCT-guided model surgical maneuvers.Translational relevanceNovel depth-based colorization and stabilization technology improves the use of intraoperative MIOCT.

Dataset Information

Integrated nonlinear optical imaging microscope for on-axis crystal detection and centering at a synchrotron beamline.

Publications

Integrated nonlinear optical imaging microscope for on-axis crystal detection and centering at a synchrotron beamline.

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